Process of dyeing protein fibers by means of insect quinones



rates PROCESS OF DYEING PROTEIN FIBERS BY MEANS OF INSECT QUINONES Rolf Karl Ladisch, Lansdowne, Pa., assignor to the United States of America as represented by the Secretary of the Army No Drawing. Application April 14, 1955 Serial No. 501,457

11 Claims. (Cl. 8-54) (Granted under Title 35, US. Code (1952), see. 266) benzoquinone, also to a process of producing ethyl-parabenzoquinone synthetically.

Accordingto Patent No. 998,370 to Meunier dated July 18, 1911, wool, silk or hair may be treated by benzoquinone either in aqueous solution or when volatilized, for the purpose of increasing the tensile strength of these fibers and enhancing their resistance to decay, heat, water and chemical agents. During this process, wool is said to assume a violet-rose color; but the patent specification is otherwise silent on the dyeing of protein fibers by benzoquinone. The performance of several synthetic quinones in the dyeing of wool has also been described in the literature (Suida, W., Z. Physiol. Chem., 85, 308,1913).

atent The invention provides not only a useful procedure for dyeing wool and other protein fibers but also a method of synthetically producing ethyl-para-benzoquinone, which is a principal quinone produced by the life process of certain flour beetles. Since insect-derived quinones are often not available, it is believed to be highly desirable to synthesize ethyl-para-benzoquinone by an inexpensive procedure. Ethyl-para-benzoquinone is needed for research, as it has been shown by the applicant (Spies, T.D., J. Am. Med. Assoc., 153, 189, 1953) that insectderived quinones, substantially consisting of ethyl-parabenzoquinone, may propagate malignant tumors in rats. However, extensive inquiry indicates that ethyl-parabenzoquinone, is not available on the market. By my invention, I have devised an inexpensive method of making ethyl-para-benzoquinone and then using this quinone, and other quinones derived from flour beetles Tribolium castaneum (Herbst) and Tribolium confusum (Duval),

for the purpose of dyeing Wool and other protein fibers,

' as well as nylon fibers also for research along the lines 7 indicated above.

A slurry consisting of 500 ml. of water, 85 ml. of concentrated sulphuric acid, and powdered Dry Ice (solid CO was prepared in a beaker kept in a Dry Ice-alcohol bath. Then 20 g. of o-ethylaniline was added to the slurry and stirred, following which 50 ml. of a 50% aqueous solution of Na Cr O was added and stirred. More powdered Dry Ice was added to produce a hard but free moving mass. The color of the mixture turned from an initial light yellow to dark brown. The beaker containing the mixture was placed in a refrigerator for 18 hours, during which period the mixture turned to a blue-black liquid mass withv a strong smell of quinone. With the mixture maintained below 5 C., 100 ml. of a 2,936,210 Patented May 10, 1960 lee 50% aqueous solution of Na Cr O was added dropwise over a period of two hours with mechanical stirring. Then 600 ml. of Z-methyl-butane was added, with stirring for one hour. At this point, a thinly viscous mass was obtained which when placed in a column and allowed to stand in a refrigerator, separated into two phases. The upper layer of Z-methyLbutane containing the quinone was siphoned oif. Crude ethylquinone in crystals formed when cooling the solvent to C. or below. These crystals were collected by fitering through a filter with a fritted glass disc, then washing with some solvent and drying in the air. The blue-black residue in the column was extracted three more times with the solvent recovered from the filtrate. The crude ethylpara-beuzoquinone was purified by sublimation in vacuo. Yield, 45% crude, 40% pure. Melting point of the pure product, 3 8.5 39.5 C.

Instead of adding Z-methyl-butane to remove the quinone, it may be removed by extracting with a solvent and evaporating the solvent, or by resorting to steam distillation, as will be understood by those skilled in the art. Such a method is covered by my co-pending application Serial No. 603,452, filed August 10, 1956, now Patent No. 2,840,571.

QUINONES AS PROTEIN FIBER DYES I Wool tops, untreated, were prepared for dyeing by four successive washings in fresh portions of chloroform, and then were dried in air.

Run N0. 1.-Twelve thousand beetles Tribolz'um confusum Duv. were collected as described in Science, 11-8, 324 (1953). One such insect contains approximately 25 micrograms of a quinone; the amount of quinone employed was therefore approximately 300 mg. The insects were finely ground in a high speed blender, together with 100 ml. of Z-methyl-butane, pure grade. The filtrate from this mixture was shaken with '12 g. of desiccant-activated silica gel to remove the quinone from the solution. After allowing the silica gel to settle, the supernatant liquid was decanted and discarded. The gel was washed three times with fresh portions of Z-methylbutane, and the remaining yellow colored gel was extracted with successive portions of boiling water. These extracts, containing the quinone, were combined (about 400 ml. resulting) and served as a dye bath for 5 g. of the wool tops prepared as described. The wool was dyed at 98 C. for 45 minutes. See the table for the re sults of this and the other runs described below.

Run N0. 2.This was like Run No. 1 except that 2 ml. of a 50% aqueous solution of acetic acid was added to the first portion of water (100 ml.) which was used to extract the quinone from the silica gel.

Run N0. 3.-In this instance, 7500 insects were processed as described in connection with Run No. 1. The dye bath was prepared as described in Run No. l, and to this solution, 710 mg. of L-arginine (free base), dissolved in a little water, was added. The bath was heated to boiling and kept at this temperature, after which it was dark red. Five grams of the wool was dyed at 98 C. for minutes in the bath.

Rune N0. 4.This run was exactly like No. 3 except that 300 mg. of glycylglycine was used instead of 'L-arginine. The bath was orange colored when immers- Was dissolved in 20 ml. of water at 40 C. and this solution was stirred into the bath containing the wool. The

Y and thoroughly washed with distilled water.

bath was slowly heated to approximately 98 C. and was kept at this temperature for 60 minutes; it turned dark red soon afer the quinone was added.

Run No. 6.A quinone solution prepared as in Run No. 1 was made up to 500 ml. of water and this solution was stirred into a solution of 300 mg. L-cysteine (free base) in 300 ml. of water. The mixture, which was completely colorless, was heated to 70 C. Then ten grams of the prepared wool was moistened with a 1% aqueous solution of hydrogen peroxide and was immersed in the bath and left for three minutes. During this brief period, both the wool and the bath became dark red-brown. The wool was withdrawn from the bath One portion of the washed Wool was then treated with a solution containing 5 ml. of concentrated ammonia in 1000 ml. of water (sample 6a); another portion was treated with a solution containing 6 ml. of a 10% solution of sulphric acid in 100 ml. of water (sample 6b). Some fresh wool was immersed in the dark red-brown dye bath, and the bath was heated to near boiling and was kept at this temperature for 120 minutes (sample 60). Run N0. 7.760 mg. of L-cysteine (free base) was dissolved in 80 ml. of Water. To this solution was added a warm solution (40 C.) of 68 mg. insect quinone in 20 ml. of water. The resulting mixture was colorless. Then 2 ml. of a 5% solution of acetic acid was added and 2 g. of the treated wool was immersed in the bath, which Was heated to 98 C. and kept at that temperature for 90 minutes.

Run No. 8.--One half pound of wheat germ was finely ground and 2000 beetles (T ribolz'um COVZfLiSMin Duv.) were cultured on the ground wheat germ for six weeks at 32 C. and 75% RH. in a dark room. Then the beetles were removed by sieving. Ten grams of the ground wheat germ was extracted successively with five portions of boiling water (100 ml. each). The decanted extracts were dark brown, yellow-brown, grey-brown, grey and light grey, in this order. The extracts were combined and concentrated on a steam bath to a volume of 150 ml. The resultant cloudy solution was centrifuged and the supernatant clear liquid was used. Four ml. of a 5% acetic acid solution was added; a clear brown solution of pH 4.5 resulted. This solution displayed an intense blue-green fluorescence in ultraviolet light. Two grams of the treated wool was immersed in the solution, heated to 98 C., and the wool was kept in the solution at this temperature for 60 minutes.

culture of T. confusum, having a grey color and a strong smell of ammonia.

Run N0. 10.The procedure of Run No. 8 was followed, except that artificially quinone-contaminated wheat germ was used, the ground wheat germ being placed in a desiccator which contained a RH. constant humidity mixture of sodium dichromate (see Langes Handbook of Chemistry, 1952, p. 1454) and enough crystalline insect quinone to keep the atmosphere within the desiccator saturated withthe vapors of the quinone. The desiccator was kept at room temperature protected from light. The wheat germ gradually became dark purple-brown and was removed after seven days, and extracted with boiling water as previously described, the extracts being dark red-brown, dark brown, dark yellow-brown, yellow-brown and light yellow-brown, in that order. The final centrifuged and acidified solution for dyeing was dark orange-brown and exhibited an intense blue-green fluorescence in ultraviolet light.

TREATMENT AFTER DYEING After the dyeing, all the wool samples were thoroughly rinsed with tap water, then excess moisture was shaken off. The stability of the color was tested by immersing a portion of the wool for fifteen minutes in a solution containing 3 ml. of concentrated ammonia in 100 ml. of water. A second portion of each wool sample was boiled in n/ 10 NaOH for a few minutes. Colors and fluorescence were noted, using a Westinghouse Purple X Lamp, 250 W.

In my pending application Serial No. 475,322, filed December 14, 1954, now Patent No. 2,827,364, I describe a process of detecting insect contamination of wheat flour by fluorescence.

ETHYL-P-BENZOQUINONE Table Run No. Dye Bath Contained Dye Bath Color W001 Dyed Extract of Wool After Experiment (11/10 NaOH) 1 Inselctfiquinone only, neutral dark brown red-browr1 yellowso u on.

2 Intsiect quinone only, acid solucolorless brown D0.

3 Insect quinonoid conjugate with deep red grey-brown Do.

L-arginine.

4 Insect quinonoid conjugate with orange. do Do.

ycyl-glycine.

5 Insect quinonoid conjugate with dark red red-brown 0- glycyl-DL-phenylalanine.

6(a) Insect quinonoid conjugatewtth dark red-brown deep red-purple. raspberry-red.

L-cysteine (free base).

do do deep blue cherry-red.

6(0) do dark red-brown (some dark brown orange-brown.

' precipitate).

7 do light pink do. dark red-purple.

8 Aq.h extract of contaminated brown light yellow-brown" dark yellow.

w ea germ.

9 Aq. extract of contaminated dark yellow-brown-.- dark yellow-brown Do.

:vhole wheat flour (old eulure 10 Aq. extract of wheat germ, yellow-brown yellow-brown Do.

treated with vapors of insect quinone.

R'un No. 9.The procedure of Run No. 8 was followed, except that whole wheat flour was employed in- All colors of samples 1 to 7 inclusive were fast when treated with dilute ammoniacal solution, as described stead of wheat germ, and the flour was from a very old above. This was also essentially true of samples 8, 9,

and 10, although traces of a yellow color eventually leached out into the dilute ammonia. Sodium hydroxide extracts of samples 8, 9 and 10 displayed an intense bluegreen fluorescence in ultraviolet light, while an intense yellow-green fluorescence was noted in the extracts of wool dyed with two of the cysteinequinone complexes (samples 6(c) and 7). The sodium hydroxide extracts of all other samples fiuorescecl with a green color. Control experiments showed that aqueous extracts from pure (insect-free) wheat germ did not dye wool.

The results summarized in the foregoing table are typical. Many additional runs too numerous to describe were carried out with dye baths containing, for example, synthetic ethyl-p-benzoquinone instead of the insect quinone, sulphuric or hydrochloric acid to acidify the bath, and amino acids such as L-histidine, L-glycine, or glycyl- L-tyrosine in those cases in which quinonoid conjugates were used for the dyeing. Many colors from light yellow and grey to pink to dark red-brown were obtained. The brown colors of quinone-dyed wool gained further in depth by after-chroming in a bath of 600 mg. potassium dichromate K Cr O- and 500 mg. of potassium bitartrate KHC H O in 800 ml. of water. An interesting sequence of colors was obtained with conjugates of cysteine and ethyl-p-benzoquinone. While the fibers remained colorless in a solution of 710 mg. L-cysteine and 400 mg. of the quinone in 400 ml. of water, they became light yellow when 1 ml. of a 50% acetic acid solution and two drops of 30% hydrogen peroxide were added to the bath. Yellow-brown wool was produced with 1 ml. of hydrogen peroxide added. Finally, a deep dark brown color was obtained on the wool from a dyeing liquid of the lastmentioned composition in which .an additional amount of cysteine (710 mg.) had been dissolved before immersing the material.

It is interesting that nylon fibers take on a rather fast pink color in a solution of 400 mg. of ethyl-p-benzoquinone in 800 ml. of water. The fibers were obtained by the processof my Patent No. 2,571,457, dated October 16, 1951, using nylon molding powder, and were dyed at 80 C. for'30 minutes.

What I claim is:

1. A process of dyeing wool which comprises making up a solution in the proportions of 80 ml. of water, 20 ml. of a neutral buffer solution, andSS mg. of glycyl-DL- phenylalanine, immersing prepared wool in said solution, adding an aqueous solution of an ethyl-parabenzoquinone-containing quinone mixture obtained from flour beetles to the bath containing the wool, heating the bath slowly to approximately 98 C. and keeping the bath at that temperature for about 60 minutes, and removing the wool, rinsing and drying.

2. A process of dyeing wool which consists in grinding flour beetles which secrete an ethyl-parabenzoquinonecontaining quinone mixture, with Z-methyl-butane, filter" ing the mixture, removing said quinone mixture from the solution so obtained by activated silica gel, washing the gel with Z-methyl-butane and then extracting it with boiling water, using the aqueous extract to form a dye bath made up to 500 ml. with water, stirring this solution into a solution of 300 mg. L-cysteine (free base) in 300 ml. of water, heating this mixture to about 70 C., moistening 10 g. of wool with a 1% aqueous solution of hydrogen peroxide and immersing the moistened wool in the bath and leaving it for approximately three minutes,

removing the wool from the bath and washing it, treating the washed wool with a solution containing 5 ml. of concentrated annnonia in 10.0 ml. of water, and finally thoroughly rinsing the wool with water and drying.

3. A process of dyeing wool which consists in grinding flour beetles which secrete an ethyl-parabenzoquinone-containing quinone mixture, with Z-methyl-butane, filtering the mixture, removing said quinone mixture from the solution so obtained by activated silica gel,

washing the gel with Z-methyl-butane and then extracting it with boiling water, using the aqueous extract to form a dye hath made up to 500 ml. with water, stirring this solution into a solution of 300 mg. L-cysteine (free base) in 300 ml. of water, heating this mixture to about 70 C., moistening 10 g. of wool with a 1% aqueous solution of hydrogen peroxide and immersing the moistened wool in the bath and leaving .it for approximately three minutes, removing the wool from the bath and washing it, and treating the washed wool with a solution of 6 ml. of a 10% solution of H in ml. of water, and finally thoroughly rinsing the wool with water and drying.

4. A process of dyeing wool which consists in grinding flour beetles which secrete an ethyl-parabenzoquinone-containing quinone mixture, with Z-methyl-butane, filtering the mixture, removing said quinone mixture from the solution so obtained by activated silica gel, washing the gel with Z-methyl-butane and then extracting it with boiling water, using the aqueous extract to form a dye hath made up to 500 ml. with water, stirring this solution into a solution of 300 mg. L-cysteine (free base) in 300 ml. of water, immersing Wool in the dye bath and heating the bath to near the boiling point and keeping it at that temperature for about minutes, and thoroughly rinsing the wool with water and drying.

5. A process of dyeing wool which comprises dissolving 760 mg. of L-cysteine (free base) in 80 m1. of water, adding a solution of 68 mg. of an ethyl-parabenzm quinone-containing quinone mixture obtained from flour beetles in 20 ml; of water, said solution having a temperature of about 40 (3., adding to the mixture 2 ml. of a 5% solution of acetic acid, immersing 2 g. of wool in the bath, heated to 98 C., maintaining the bath at that temperature for about 90 minutes, and thoroughly rinsing the wool with water and drying.

6. A process of dyeing a member of the group consisting of protein fibers and nylon fibers which comprises exposing the fibers to a quinonoid conjugate being the reaction product of an amino acid with. a member of the group consisting of synthetic ethyl-parabenzoquinone and an ethyl-parabenzoquinone-containing quinone mixture obtained from flour beetles, in the presence of moisture and at temperatures ranging from room temperature to about 98 C. for a suificient time to change the color of the protein fibers to the desired hue, removing the fibers, rinsing and drying.

7. A process as claimed in claim 6, wherein said quinonoid conjugate is a conjugated quinonoid of cereal protein.

8. Process according to claim 6, wherein said amino acid is arginine.

9. Process according to claim 6, wherein said amino acid is glycyl-glycine.

10. Process according to claim 6, wherein said amino acid is glycyl phenyl-alanine.

11. Process according to claim 6, wherein said amino acid is cysteine.

References Cited in the file of this patent The Chem. of Tanning Processes, Publrs., New York, 1956, p. 285.

Hoppe-Seylers Zeitschrift fiir physiologische Chemie, vol. 85, for 1913, pp. 317-323 (art. by W. Suida).

by Gustavson,

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No, 2,936,210 May 10, 1960 Rolf Karl Ladisch It is herebfi certified that error appears in the-printed specification of the above numbered patent requiring c orrection and that the said Letters Patent should read as corrected below.

Column 5, line 68, for "10.0 ml." read 1000 ml.

Signed and sealed this 11th day of October 1960.

(SEAL) Attest:

KARL H. AXLINE ROBERT C.. WATSON Attesting Officer Commissioner of Patents 

1. A PROCESS OF DYEING WOOL WHICH COMPRISES MAKING UP A SOLUTION IN THE PROPORTIONS OF 80 ML. OF WATER, 20 ML. OF A NEUTRAL BUFFER SOLUTION, AND 55 MG. OF GLYCLY-DLPHENYLALANINE, IMMERSING PREPARED WOOL IN SAID SOLUTION, ADDING AN APUEOUS SOLUTION OF AN ETHYL-PARZBENZOQUINONE-CONTAINING QUINONE MIXTURE OBTAINED FROM FLOUR BEETLES TO THE BATH CONTAINING THE WOLL, HEATING THE BATH SLOWLY TO APPROXIMATELY 98*C. AND KEEPING THE BATH AT THAT TEMPERATURE FOR ABOUT 60 MINUTES, AND REMOVING THE WOOL, RINSING DRYING. 